Pratt Stephen J P, Hernández-Ochoa Erick O, Lee Rachel M, Ory Eleanor C, Lyons James S, Joca Humberto C, Johnson Ashley, Thompson Keyata, Bailey Patrick, Lee Cornell J, Mathias Trevor, Vitolo Michele I, Trudeau Matt, Stains Joseph P, Ward Christopher W, Schneider Martin F, Martin Stuart S
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD, USA.
Department of Physiology, University of Maryland School of Medicine, Baltimore, MD, USA.
Oncotarget. 2018 May 18;9(38):25008-25024. doi: 10.18632/oncotarget.25186.
Aggressive cellular phenotypes such as uncontrolled proliferation and increased migration capacity engender cellular transformation, malignancy and metastasis. While genetic mutations are undisputed drivers of cancer initiation and progression, it is increasingly accepted that external factors are also playing a major role. Two recently studied modulators of breast cancer are changes in the cellular mechanical microenvironment and alterations in calcium homeostasis. While many studies investigate these factors separately in breast cancer cells, very few do so in combination. This current work sets a foundation to explore mechano-calcium relationships driving malignant progression in breast cancer. Utilizing real-time imaging of an scratch assay, we were able to resolve mechanically-sensitive calcium signaling in human breast cancer cells. We observed rapid initiation of intracellular calcium elevations within seconds in cells at the immediate wound edge, followed by a time-dependent increase in calcium in cells at distances up to 500μm from the scratch wound. Calcium signaling to neighboring cells away from the wound edge returned to baseline within seconds. Calcium elevations at the wound edge however, persisted for up to 50 minutes. Rigorous quantification showed that extracellular calcium was necessary for persistent calcium elevation at the wound edge, but intercellular signal propagation was dependent on internal calcium stores. In addition, intercellular signaling required extracellular ATP and activation of P2Y receptors. Through comparison of scratch-induced signaling from multiple cell lines, we report drastic reductions in response from aggressively tumorigenic and metastatic cells. The real-time scratch assay established here provides quantitative data on the molecular mechanisms that support rapid scratch-induced calcium signaling in breast cancer cells. These mechanisms now provide a clear framework for investigating which short-term calcium signals promote long-term changes in cancer cell biology.
侵袭性细胞表型，如不受控制的增殖和迁移能力增强，会导致细胞转化、恶变和转移。虽然基因突变无疑是癌症发生和发展的驱动因素，但越来越多的人认为外部因素也起着重要作用。最近研究的两种乳腺癌调节因子是细胞机械微环境的变化和钙稳态的改变。虽然许多研究分别在乳腺癌细胞中研究这些因素，但很少有研究将它们结合起来进行研究。这项工作为探索驱动乳腺癌恶性进展的机械钙关系奠定了基础。利用划痕试验的实时成像，我们能够解析人乳腺癌细胞中机械敏感的钙信号。我们观察到，在紧邻伤口边缘的细胞中，细胞内钙在几秒钟内迅速升高，随后在距离划痕伤口达500μm的细胞中，钙随时间依赖性增加。向远离伤口边缘的相邻细胞的钙信号在几秒钟内恢复到基线。然而，伤口边缘的钙升高持续了长达50分钟。严格的定量分析表明，细胞外钙是伤口边缘钙持续升高所必需的，但细胞间信号传播依赖于细胞内钙库。此外，细胞间信号传导需要细胞外ATP和P2Y受体的激活。通过比较多种细胞系的划痕诱导信号，我们报告了具有高度致瘤性和转移性的细胞的反应大幅降低。这里建立的实时划痕试验提供了关于支持乳腺癌细胞中快速划痕诱导钙信号的分子机制的定量数据。这些机制现在为研究哪些短期钙信号促进癌细胞生物学的长期变化提供了一个清晰的框架。
